Poly(A) polymerase from Escherichia coli adenylylates the 3′‐hydroxyl residue of nucleosides, nucleoside 5′‐phosphates and nucleoside(5′)oligophospho(5′)nucleosides (Np_nN)

Abstract

The capacity of Escherichia coli poly(A) polymerase to adenylylate the 3′‐OH residue of a variety of nucleosides, nucleoside 5′‐phosphates and dinucleotides of the type nucleoside(5′)oligophospho(5′)nucleoside is described here for the first time. Using micromolar concentrations of [α‐32P]ATP, the following nucleosides/nucleotides were found to be substrates of the reaction: guanosine, AMP, CMP, GMP, IMP, GDP, CTP, dGTP, GTP, XTP, adenosine(5′)diphospho(5′)adenosine (Ap2A), adenosine (5′)triphospho(5′)adenosine (Ap3A), adenosine(5′)tetraphospho(5′)adenosine (Ap4A), adenosine(5′)pentaphospho(5′)adenosine (Ap5A), guanosine(5′)diphospho(5′) guanosine (Gp2G), guanosine(5′)triphospho(5′)guanosine (Gp3G), guanosine(5′)tetraphospho(5′)guanosine (Gp4G), and guanosine(5′)pentaphospho(5′)guanosine (Gp5G). The synthesized products were analysed by TLC or HPLC and characterized by their UV spectra, and by treatment with alkaline phosphatase and snake venom phosphodiesterase. The presence of 1 mm GMP inhibited competitively the polyadenylylation of tRNA. We hypothesize that the type of methods used to measure polyadenylation of RNA is the reason why this novel property of E. coli poly(A) polymerase has not been observed previously.

Polyphosphates strongly inhibit the tRNA dependent synthesis of poly(A) catalyzed by poly(A) polymerase from Saccharomyces cerevisiae

“…The first, but not the second enzyme, adenylylates the 3′‐hydroxyl residues of nucleosides, nucleoside 5′‐phosphates and nucleoside(5′)oligophospho(5′)nucleosides or dinucleoside polyphosphates (Np n N). Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by the yeast enzyme, activation not observed using the enzyme from E. coli [1]. In the absence of primer, the velocity of the enzyme from S. cerevisiae towards its substrate ATP displayed sigmoidal kinetics with a Hill coefficient of around 1.6 and a K m ( S 0.5 ) value of around 0.3 mM.…”

“…We have recently described novel properties for the poly(A) polymerases from Escherichia coli [1] and from Saccharomyces cerevisiae [2]. The first, but not the second enzyme, adenylylates the 3′‐hydroxyl residues of nucleosides, nucleoside 5′‐phosphates and nucleoside(5′)oligophospho(5′)nucleosides or dinucleoside polyphosphates (Np n N).…”

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

“…At 30 min and 2 h, 10 l of the parasite mixture was centrifuged at 13,000 rpm in an Eppendorf Centrifuge 5415D, the supernatant was removed, and the parasite pellet was resuspended in 5 l of glacial acetic acid to lyse the cells and terminate the reaction (46). The lysate was spotted onto a PE-SIL-G TLC plate with fluorescent indicator (GE Healthcare), so that nonradioactive standards could be detected, and developed in dioxane/ammonium hydroxide/water at a ratio of 6:1:5 (v/v/v) (47). The TLC plate was exposed to X-ray film at Ϫ80°C and developed in a standard X-ray film developer.…”

A comparative study of microbial diversity and community structure in marine sediments using poly(A) tailing and reverse transcription-PCR

“…The approaches are summarized in Figure1 . Since the reaction buffer composition and source of poly(A) polymerase can reportedly affect the efficiency of the poly(A) tailing reaction (Raynal and Carpousis, 1999;Sillero et al, 2001), we used two commercially available poly(A) polymerases: Escherichia coli poly(A) polymerase (New England BioLabs, hereafter denoted as NEB) and Takara Bio poly(A) polymerase. Each poly(A) tailing reaction was conducted in 20 μl of reaction mixture containing 10 μl of purified 16S rRNA solution.…”